Tissue Lysate Preparation Protocol

1. Weigh tissue sample in a 50 mL tube.

2. While keeping sample on ice, wash with cold 1X PBS and aspirate off PBS.

3. Repeat until wash buffer appears clear.

4. Add sufficient volume of cold lysis buffer to cover sample (about 3 times the weight of sample in volume; i.e. 500 mg sample will receive 1.5 mL lysis buffer)

5. Grind/homogenize tissue in tube and incubate on ice for at least one hour.

6. Transfer mixture to microcentrifuge tubes and spin at 14,000 rpm for 30 min at 4°C.

7. Poke through lipid layer and remove supernatant (this is the lysate). Throw away cellular debris and lipids.

8. If necessary, respin supernatant at14,000 rpm and repeat step 7 to obtain clean lysate free of lipid and debris.

9. Determine protein concentration using Bradford Protein Assay.

10. Store at -80°C until ready to work up.

11. Avoid freeze/thawing to protein as much as possible.

5X Lysis buffer composition (stored in 4°C)

- 50 mM Tris pH 7.5

- 650 mM NaCl

- 5 % Triton-X100

- 50 mM NaF

- 50 mM NaPi pH 7.5 (sodium phosphate)

- 50 mM NaPPi pH 7.5 (sodium pyrophosphate)

These are added right before use

- 0.02 mg/mL Rnase

- 0.2 mg/mL Dnase

- 1 mM PMSF

- 1X Protease inhibitor cocktail (PIC)

 

100X PIC

- 1.6 mg/mL Benzamidine HCl

- 1.0 mg/mL Phenanthroline

- 1.0 mg/mL Aprotonin

- 1.0 mg/mL Leupeptin

- 1.0 mg/mL Pepstatin A

 

dissolve in 100% ETOH and stored at -20°C